The present invention relates generally to microscopy and more specifically to an improved confocal microscope.
The art of microscopy deals with the development of methods and instruments for magnifying. A significant portion of microscopy deals with using light from the visible portion of the spectrum to illuminate the sample to be magnified. This aspect of the art of microscopy is known as light microscopy.
It is well understood that many factors influence the maximum axial and lateral resolution attainable using light microscopy techniques. One of these limiting factors is the field size. Specifically, when the field of view of a lens is extremely small, the axial and lateral resolution of the image being magnified can in fact be greater than when the field of view of the lens is broad (or not limited). These theoretical considerations set the stage for the development of an apparatus known as the confocal microscope.
In U.S. Pat. No. 3,013,467 (hereinafter '467) issued on Dec. 19, 1961 to M. Minsky (see FIG. 1), a confocal microscope is disclosed. In this patent, Minsky discloses specimen 22 mounted upon the reflective surface of mirror 15. A beam-splitting plate 17 is interposed between the collimating plate or wall 14 and lens 11. The reflective surface of beam-splitting plate 17 faces lens 11, while the transparent surface of plate 17 faces pin hole aperture 16.
The light reflected from bulb 10 by reflector 12 is collimated by pin hole aperture 16 of plate 14 to provide a point source of light A. Divergent beam B,B passes through beam-splitting plate 17 and then through lens 11 becoming convergent beam C,C. Focal point D of beam C,C is located on specimen 22 and becomes divergent beam E,E which is reflected from mirror 15 back through lens 11. Lens 11 forms convergent beam F,F which is reflected perpendicularly from beam-splitting plate 17 as indicated by beams F',F' which converge to their focal point G at pin hole aperture 26 of plate 24. Photoelectric cell 28 is located in alignment with aperture 26 to measure the intensity of the light passing therethrough. Because pin hole apertures 16 and 26 lie upon the optical axis OA of the confocal microscope of FIG. 1, the point source of light A and the specimen point of illumination D both originate on optical axis OA, while the point image G terminates on the optical axis OA. Therefore, all of the light rays accepted by photoelectric cell 28 must pass through specimen 22 at point D an optical axis OA and pass again through optical axis OA at point G. Light scattered from points other than point D of specimen 22 is, for the most part, rejected from the optical system. Such scattered rays may pass through and be refracted by lens 11 but will not be directed to pin hole aperture 26. Rather, this scattered light will strike the body of plate 24 and be rejected from the optical system. Such rays can re-enter the optical system only by again being scattered, and the possibility of this scattering taking place along a line through point D on optical axis OA is remote. Pin hole aperture 26 increases the optical resolution of the system by its action of squaring the intensity pattern distribution of the image defraction.
Because the confocal microscope of FIG. 1 provides a high degree of selectivity, the following advantages are gained:
(1) minimum image blurring,
(2) increase in signal-to-noise ratio,
(3) increase in effective resolution,
(4) high resolution light microscopy through unusually thick and highly scattering specimens, and
(5) very narrow depth of focus.
As was discussed above in conjunction with the prior art confocal microscope of FIG. 1, the optical system disclosed therein brings into focus the light originating at a single point on the optical axis. Thus, if one desires to look at several portions of sample 22, some means must be provided to move the point of specimen illumination D. Minsky discloses one such means in his '467 patent which involves continually moving (or scanning) sample 22 relative to the optical system. Although this technique works satisfactorily for small samples, larger samples cannot be easily adapted to this type of scanning and alternative methods are used.
One alternative to the above-mentioned method of moving the sample relative to the optical system, involves the technique of moving (or scanning) specimen illumination point D relative to sample 22. Although several different techniques may be used to accomplish the scanning of illumination point D, a popular technique involves using a spinning opaque wheel 90 (also known as a Nipkow disk) that is perforated by a series of pin holes 92 (see FIG. 2A). The successive holes are placed at a constant angle apart 94 around the center of disk 90 but on a constantly decreasing radius 96 (i.e. arranged as an Archimedes spiral). The basic idea behind the Nipkow disk is instead of using a single pin hole 16 in plate 14 and moving sample 22, a large number of pin holes are placed in plate 14 thereby providing a means of scanning specimen illumination point D while maintaining specimen 22 stationary. Pin holes 92 are sufficiently separated so that there is no interaction between the images formed by the individual pin holes. The complete image is formed by moving the pin holes so as to fill the space in between them. Typically, moving the pin holes involves rotating disk 90 about its center. The pin hole arrangement seen in the prior art Nipkow disk of FIG. 2A would produce a raster scan pattern across sample 22 as depicted in FIG. 2B.
Although the basic concepts of confocal microscopy are understood and documented (for example see generally Handbook of Biological Confocal Microscopy, edited by James B. Pawley, Integrated Microscopy Resource for Biomedical Research University of Wisconsin-Madison, Madison, Wisconsin, revised edition, Plenum Press, New York and London, specifically see Chapter 1, Foundations of Confocal Scanned Imaging in Light Microscopy; Chapter 10, Intermediate Optics in Nipkow Disk Microscopes; and Chapter 11, The Role of the Pin Hole in Confocal Imaging Systems, also see Physics Today, September 1989, by Gordon S. Kino of Stanford University), current designs continue to be improved upon. For example, notwithstanding the above-mentioned advantages associated with using a Nipkow disk to scan the light source across the sample, a major disadvantage is that the Nipkow disk blocks typically 99% of the illuminating light requiring the use of a very intense light source (such as an arc lamp or laser). In addition to the above drawback, the use of Nipkow disks also produces a high percentage of reflected light (light which does not pass through the pin hole opening of the disk) which, in turn, causes artifacts in the final image.
Accordingly, it is an object of this invention to provide a Nipkow disk scanning system which has an improved transfer efficiency between the light source and the specimen point of illumination.
It is a further advantage of this invention to provide an improved optical scanning system which reduces the percentage of reflected light thereby reducing errors due to artifacts.
It is still a further advantage of this invention to provide an optic system for use on a confocal microscope employing Nipkow disk scanning which offers superior transfer efficiency between the illumination source and the sample being illuminated.